Electrospray ionization mass spectrometry (ESI MS) has become an indispensable tool in the study of protein higher order structure and dynamics, as it allows macromolecular properties to be probed under a variety of conditions, including analysis of non-native conformations of transient species, such as protein folding intermediates. In recent years, ESI MS was also applied to study protein aggregation, although such studies are typically limited to the analysis of various species that are populated at the end-point of the aggregation process. The ability of ESI MS to provide information on biopolymer complexes in real time has been used to monitor ordered dissociation and assembly of protein oligomers; however, detailed characterization of the aggregation process has remained out of reach of conventional ESI MS.
The transient nature of protein aggregation is an inherent feature of the process that makes it very difficult to provide an accurate description of this process. Once the aggregation process is initiated, it proceeds very fast, making it extremely difficult to produce distinct in process snap-shots. Even though ESI MS provides an elegant way to obtain snap-shots of various dynamic processes, a feature that has been used to study phenomena ranging from protein interaction with their endogenous ligands to enzyme catalysis, straightforward application of ESI MS to on-line monitoring of aggregation processes is impractical due to the unfavorable time scale of the processes. Therefore, it is not surprising that most studies of protein aggregation focus on the endpoints of this process, i.e., early precursors to aggregation and the high molecular weight oligomers.
Another factor that greatly complicates the analysis of the aggregation process is the astonishing polymorphism exhibited by both the products and the intermediate states of the aggregation process. Earlier attempts to reduce the complexity of these ensembles by using a combination of separation techniques (e.g., size-exclusion chromatography, SEC) and ESI MS were only partially successful, since the intermediate oligomeric species collected as SEC fractions are very dynamic and frequently continue to evolve after (and perhaps even during) the separation process. As a result, a detailed mechanistic understanding of protein aggregation is still lacking, and the role of various non-native protein states in this process remains a subject of on-going debates.
Protein aggregation can be modulated in vitro by a variety of factors, and protein solution temperature is one of the parameters that have a profound effect on this process. The effect of heat on protein behavior is widely exploited in biotechnology, e.g., in accelerated stability studies, where products are stressed by exposing them to high temperatures. However, dramatic changes in physical and chemical properties that frequently occur during stress-testing of proteins are difficult to interpret, which makes meaningful analysis extremely difficult. The capability of ESI MS to explore both conformation and binding properties of biopolymers makes this technique an attractive tool for characterizing protein behavior under heat-stress conditions. Nonetheless, even though the idea to monitor protein behavior in solution as a function of temperature using ESI MS was articulated nearly two decades ago, very few examples of using ESI MS to study protein behavior as a function of temperature have been reported so far.
An improved apparatus and methods are needed to probe fast, dynamic processes such as protein aggregation.